This present invention relates to culture vessels, sometimes referred to as bioreactors, for growing cells, three dimensional cellular aggregates, xe2x80x9corganoids,xe2x80x9d tissues or the like (at times referred to below as xe2x80x9cparticlesxe2x80x9d) and their method of use for carrying out various cellular processes with tissues and/or organoids and for diagnostic testing and research.
This invention relates to improvements in culture vessels for growing or culturing cells, cellular aggregates, tissues, organoids and the like and to methods for using the same, and in particular to improvements in culture vessels as disclosed in U.S. Pat. Nos. 5,989,913 and 6,080,581, issued to Charles D. Anderson on Nov. 23, 1999 and Jun. 27, 2000, respectively, both hereby incorporated herein by reference in their entirety. Both of these patents are not admitted to be prior art with respect to the present invention by their incorporation into this application.
Most prior art culture vessels have been cylindrical in shape with flat, vertical interior end walls and many prior art vessels incorporate other interior obstructions, such as central cylindrical cores, agitators or impellers, which also impact the flow paths of cells, cellular aggregates, xe2x80x9corganoidsxe2x80x9d or tissue particles (see definition within), and are thought to create impact and/or abrasion surfaces impeding toroidal flow. U.S. Pat. No. 6,001,642 to Tsao, entitled xe2x80x9cBioreactor and Cell culturing Process Using the Bioreactor,xe2x80x9d discloses an asymmetrical bioreactor having one spherically-shaped wall facing and connecting to a second flat wall. This bioreactor is commonly named the Hydrodynamic Focusing Bioreactor. The center of the flat wall features a spinner type agitator that projects into the middle of the culture chamber and is designed to enhance bubble removal. The bioreactor of the ""642 patent does not have a chamber defined by a smooth, non-flat, spherical, oblate spherical or curved wall that is symmetrical about an axis in that one wall of the culture chamber is essentially flat except for a centrally mounted agitator. The front and back walls of the ""642 bioreactor join at a very sharp angle that would impede toroidal flow. There is an agitator in the center of the culture chamber of the ""642 bioreactor that impedes toroidal flow across the center of the vessel and creates turbulence. Finally, the hydrodynamic focusing bioreactor is not intended to facilitate toroidal flow but is intended to focus the flow within the vessel into a xe2x80x9cdonutxe2x80x9d pattern. The focusing action created by this centrally located spinner agitator causes shear, turbulence, and vessel wall impacts especially against the flat back wall of the device.
The need exists for a continuously renewed culture vessel, rotated over an essentially horizontal axis, that minimizes interior structures that impedes the ability of cells, cellular aggregates or tissues to freely expand in three dimensional growth under minimally disturbed toroidal flow. The vessel would have an inlet and an outlet in fluid communication with a pump and at least one filter in fluid communication with an outlet. There is a need for a version of such a culture vessel or bioreactor that may be operated as a batch reactor. There is also a need for a method of use of such culture vessels or bioreactors for the production of biological products, and for the removal of toxins and biological waste from a fluid using cellular mechanism. The culture vessels or bioreactors of the present invention address these needs.
The present invention pertains to a culture vessel essentially horizontally rotatable on a horizontal axis, the vessel having at least one chamber. The chamber in one preferred embodiment has spherical, oblate, extended spherical or extended oblate spherical wall portions. Typically, though not necessarily, the chamber would be symmetrical about the longitudinal axis. The chamber has an inlet and outlet in fluid communication with at least one pump. At least one filter in the chamber passes fluid and cellular waste out the outlet while retaining the cells, tissue, organoids or the like. Also the invention relates to methods for using such a culture vessel for growing cells, cellular aggregates, organs, tissues and the like and for the production of biological products or the removing of toxins or biological waste from a fluid using cellular mechanisms.
Testing has confirmed that a culture vessel with a continuously renewing fluid medium therethrough need not be cylindrical, as is the custom in the industry. Testing shows that the chamber is not limited to a cylindrical shape, but a spherical or oblate spherical or extended spherical or extended oblate spherical shape may provide an improved shape for a culture chamber. A spherical or oblate spherical or extended spherical or extended oblate spherical shape for a chamber offers, as viewed at in cross section through a longitudinal axis, rounded corners that facilitate growth or other operative cultures by minimizing impact forces and shearing forces upon particles moving within the chambers. Such lateral movement is described below. It has further been determined that in the simplest embodiments of a culture chamber, only one filter is needed, in fluid communication with an outlet. A chamber can function adequately without the safety feature of a second upstream filter, although the ability to reverse flow between a chamber inlet and outlet would be sacrificed. Upstream valving could be employed, in lieu of an upstream filter to protect against cell migration through an inlet.
As disclosed in the referenced and incorporated patents above, particles continuously descend or sediment through a culture fluid in a vessel horizontally rotated about a longitudinal axis. While it had been thought that particles in such circumstances descend in a linear or two-dimensional flow path and were uniformly distributed in the horizontally rotated culture vessel, it has been observed that distinct or visible bands of particles are formed in such rotated vessels. The flow path is three-dimensional. Particles migrate along toroidal, three dimensional flow paths. Surmise is that such toroidal flow may be due in part to Coriolis forces acting on the particles as they move inside of the rotated vessel.
The phrase, xe2x80x9ctoroidal flowxe2x80x9d indicates that the particles do not simply rotate in a simple circular pattern with the chamber fluid about an essentially horizontal longitudinal chamber axis. If the end point of a particle at the end of a full 360 degree rotation about a longitudinal axis could be plotted, the following would be apparently detected. A cross section taken through the longitudinal axis would show the end point migrating in a small circle over time, in the plane of the cross section, traversing a portion of the circumference of that circle with each 360 degree revolution around the horizontal axis. A cross section taken normal to the longitudinal axis would show the end point migrating in another small circle in the plane of the cross section, traversing a segment of the circumference of that circle, with each 360 degree rotation. The path of a particle would appear to describe a three dimensional sphere or egg shape over time.
Growth of cells, cellular aggregates, organoids or tissues and the like may be increased and enhanced to the extent the cells, cellular aggregates, organoids or tissues are permitted to freely expand in three dimensions while remaining undisturbed in toroidal flow ,as described above.
Observation of particle motion in horizontally rotated bioreactors indicates that natural toroidal flow is disturbed when the particles collide or have impact with interior walls (and/or any other obstructions within a vessel). The violence of the impact affects growth. Reduced shearing forces and/or reduced number of impacts with interior walls facilitates undisturbed motion and results in faster cellular growth, larger sized cellular aggregates or tissues, more efficient removal of toxins and biological wastes and the enhanced secretion of desired bioproducts.
The geometry of the interior of a rotated culture vessel can be a factor in decreasing the number of vessel wall impacts and/or shearing forces of impact. Culture vessels having spherical, oblate spherical, extended spherical and/or extended oblate spherical wall portions or viewed by cross section through a longitudinal axis, are conducive to minimizing shearing forces and/or the number of vessel wall impacts. A reduced angle of impact should result in lower shearing forces. Lower shearing forces should result in less transferred momentum. Reduced shearing forces and number of impacts by culture vessels having spherical, oblate spherical, extended spherical and/or extended oblate spherical wall portions thus should cause less of a departure from normal toroidal flow for growing cells, cellular aggregates and tissues. In fact, vessels having spherical, oblate spherical, extended spherical or extended oblate spherical walls have been shown to produce faster cell growth and to produce larger size aggregates or tissues than what has been achieved in prior art vessels having substantial flat interior end walls or surfaces than what has been achieved in prior art vessels having substantially flat interior end walls or surfaces and cylindrical bodies.
The present invention is directed to a horizontally rotatable culture vessel or bioreactor that has smooth, curved, spherical, oblate spherical, non-flat walls for growing cells, cellular aggregates, tissue particles or xe2x80x9corganoidsxe2x80x9d and a method for use of such vessel or bioreactor. A preferred embodiment of the invention is a culture vessel which includes a housing having a chamber defined by a spherical, oblate spherical or curved wall symmetrical about an axis, an inlet and an outlet. One embodiment of such a vessel also includes at least one filter in fluid communication with at least one of the inlets and outlets, the filter structured to pass culture media and cellular waste and to retain cells, cellular aggregates or tissues and a means for rotating the culture vessel about its proximate horizontal axis. This filter version will include a pump in fluid communication with the inlet or outlet, to flow the culture media through the vessel and a method of transferring dissolved gases into and out of the culture media. The gas exchange device will be exterior to the culture vessel but will be in fluid communication with the culture vessel. The vessel or bioreactor will be rotated about a substantially horizontal axis.
The other version of the spherical, oblate spherical, extended spherical, extended oblate spherical or curved wall vessel will be a batch culture device. The batch culture device will have a gas permeable membrane within the housing. In some embodiments, at least part of the wall of this vessel will be made of a gas permeable material. The culture media in the batch culture device will be changed by stopping the rotation of the vessel and removing and replacing a given amount of culture medium as a batch. Typically approximately ⅓ culture medium is retained and is known in the art as xe2x80x9cconditioned mediumxe2x80x9d, approximately ⅔ of fresh medium is added. After the culture medium has been changed in this manner, rotation will be resumed. The gas permeable batch culture device is a low cost, short duration alternative to the continuous perfused system. This system may be used for chemotherapy testing, perhaps 5 to 10 different vessels would have cells grown with cancer cells taken from the patient. A physician may then test various modalities of treatment on the different batch vessels to determine which would have the greatest measure of success in the patient. This system could be built as a disposable or reusable device.
The culture vessel or bioreactor of the present invention may be used to grow cells, cellular aggregates, tissue or tissue xe2x80x9corganoidsxe2x80x9d in a continuous manner or versions of the invention will operate as batch culture devices. In the batch culture version, at least part of the smooth, curved, spherical, oblate spherical, non-flat vessel wall will be constructed of gas permeable material.
The filter or filters of the perfused culture vessel or bioreactor of the present invention may be disposed about a chamber of the housing in one or more preferred embodiments. A filter or filters may be located proximate an axis of rotation, or an inlet or outlet or both. The filter or filters may be disposed about at least one chamber defined by a curved wall. In addition, a filter may enclose cells, cellular aggregates or tissues in at least one chamber of the housing. The filter or filters may be structured to pass culture media and waste from an inlet while retaining cells, cellular aggregates, xe2x80x9corganoidsxe2x80x9d or tissues. The filter may also be structured to pass culture media from the outlet to the inlet while retaining such cells.
In any of the preferred embodiments of the culture vessel or bioreactor of this invention, at least a portion of the chamber may have a substantially curved, ellipsoid, oblate ellipsoid, spherical, extended spherical, oblate spherical or extended oblate spherical shape. Further, one or more of the preferred embodiments of this invention may include means for removing bubbles from the media such as a recession for trapping bubbles in the chamber and a port for releasing the trapped bubbles. Also, a gas exchange device and means for monitoring temperature, pressure or PH of the chamber may be included. When the vessel is operated in a continues manner, means for monitoring the culture media flow rate may also be included.
Another embodiment of the invention is a method for growing or maintaining cells, cellular aggregates or tissue particles or xe2x80x9corganoidsxe2x80x9d. The method includes introducing living cells, cellular aggregates, xe2x80x9corganoidsxe2x80x9d or tissue material into a culture media in a vessel chamber having a spherical or oblate spherical or continuos curved wall symmetrically about an axis, an inlet and an outlet. In the perfusion version of the invention the chamber has at least one filter, typically two filters, an inlet filter and an outlet filter in fluid communication with the inlet or outlet and structured to pass culture media and cellular waste and to retain cells, cellular aggregates or tissues. The cells, cellular aggregates or tissues are suspended by rotating the vessel about its horizontal axis. The method may further include reversing the flow of the culture medium through the vessel in a continuos manner to use the outlet filter as an inlet to unclog and remove debris from the outlet filter and to increase filter life by using the inlet filter as an outlet filter. A typical type of debris would be material that clogs filters such as mucin produced by the cells, cellular aggregates, xe2x80x9corganoidsxe2x80x9d or tissue materials. In the batch culture version at least part of the surface of the vessel is made up of gas permeable material so that the cells, cellular aggregates, xe2x80x9corganoidsxe2x80x9d or tissue materials are provided dissolved oxygen and the metabolic dissolved carbon dioxide is removed from the culture vessel. Expended media is removed from the batch culture vessels by stopping the vessel, withdrawing a given amount of expended media and replacing it with a fresh batch of media. This process was explained in more detail above. In order to maintain a sterile environment, these procedures are performed under a sterile hood, which maintains a clean environment.
Either the perfused or the batch culture version may be used to produce biological products from the growth or maintenance of cells or tissues. An example of such a method of use would be the production of human hormones, enzymes or protein pharmaceuticals.
Still another embodiment of this invention is a method for producing desired biological products into or removing toxins or biological waste material from a circulating fluid using cellular mechanisms. The method includes establishing a culture of live cells or organoids in a culture fluid in a chamber that has a spherical or oblate spherical; or continuous curved wall symmetrically about an axis. Fluid containing waste material is added to the vessel containing the culture of cells, cellular aggregates, xe2x80x9corganoidsxe2x80x9d or tissue materials, having a chamber defined by as spherical, oblate or extended curved wall symmetrical about an axis, an inlet and an outlet. Oxygen and nutrients are provided to the organoids. The cells or organoids are suspended by rotating the vessels about its horizontal axis. Multiple vessels may be connected in series. When the first vessel in the series becomes spent, it is removed and a fresh vessel is added to the other end of the series. The toxins or waste materials are removed from the fluid using the cellular metabolic mechanisms of the organoids. An example of such a device would be a xe2x80x9cLiver Assist Devicexe2x80x9d which would use liver cells or tissue cultures in this invention to detoxify blood plasma that would be reinfused into the patient. An example of the use of the invention to produce a desired biological product would use pancreatic islets to produce insulin into the patients plasma that would be reinfused into the patient.